EpiBlot: Joint Mapping of Chromatin Accessibility and Targeted Proteomics in HER2-expressing Breast Cancer Systems

HER2 proteoforms promote therapeutic resistance and aggressiveness in HER2-positive breast cancer, yet their epigenetic consequences remain poorly defined. Here, we establish EpiBlot, a joint assay incorporating a customized plateATAC-seq workflow that minimizes sample inputs with single-cell western blotting to concurrently profile chromatin accessibility with protein and proteoform expression. We applied our method to engineered MCF7 cells expressing HER2 proteoforms - full-length p185HER2 or truncated 611-CTF -, where we evaluated the impact of such proteoforms on the epigenetic and protein profiles after lapatinib or doxorubicin exposure. Expression of 611-CTF elicits pervasive chromatin remodeling, whereas p185HER2 provokes only modest accessibility shifts under the same treatments. EpiBlot reveals that treatment with doxorubicin drives extensive genome-wide accessibility changes, while lapatinib treatment produces limited global effects but unmasks proteoform-specific responses. Concordance between chromatin accessibility and protein abundance is moderate, underscoring complex regulatory coupling. Extending this dual-modality approach to HER2-low patient-derived organoids uncovers distinct chromatin states and reveals a subpopulation of triple-negative breast-cancer cells expressing truncated HER2 proteoforms. We anticipate that EpiBlot will highlight the value of multimodal profiling with proteoform identification for dissecting tumor heterogeneity and therapeutic response in cancer.

HER2蛋白亚型（HER2 proteoforms）在HER2阳性乳腺癌中可促进治疗耐药性与肿瘤侵袭性，但其相关表观遗传后果迄今仍未得到充分阐释。本研究构建了EpiBlot检测技术：一种整合定制化板基转座酶可及性测序（plateATAC-seq）流程的联合检测方法，该流程可在最小化样本输入量的同时，结合单细胞蛋白质印迹技术（single-cell western blotting）同步分析染色质开放状态与蛋白质及蛋白亚型的表达水平。我们将该方法应用于两类工程化MCF7细胞——分别表达全长p185HER2或截短型611-CTF——，并评估了此类蛋白亚型在经拉帕替尼（lapatinib）或多柔比星（doxorubicin）处理后，对细胞表观遗传谱与蛋白质组谱的影响。实验结果显示，611-CTF的表达会引发广泛的全基因组染色质重塑，而在相同药物处理条件下，p185HER2仅能引发轻微的染色质开放状态变化。EpiBlot技术揭示，多柔比星处理可驱动大范围的全基因组染色质开放状态改变，而拉帕替尼处理仅产生有限的全局效应，但可精准揭示蛋白亚型特异性的细胞应答反应。染色质开放状态与蛋白质丰度之间的相关性中等，这凸显了二者之间调控耦合关系的复杂性。将这种双模态分析方法拓展应用至HER2低表达患者来源类器官（patient-derived organoids），可揭示独特的染色质状态，并发现一群表达截短型HER2蛋白亚型的三阴性乳腺癌（triple-negative breast cancer）细胞亚群。我们预期，EpiBlot技术将凸显结合蛋白亚型鉴定的多模态谱分析在解析肿瘤异质性与癌症治疗应答中的应用价值。